Development of an Accurate and Robust Polarizable Molecular Mechanics Force Field from ab Initio Quantum Chemistry

We have produced a polarizable force field for a series of small molecules, representative of functional groups found in organic and biochemical systems. We have used high-level ab initio results for fitting values of all the parameters except for the dispersion-term coefficient B in the −B/r6 energy term, which, although obtained from comparison with experimental condensed-phase data, depended only on atomic number of the site in hand. Heats of vaporization and densities of the pure liquids, computed with molecular dynamics, agreed with experiment within ca. 0.5 kcal/mol and 5%, respectively

Effects of Restrained Sampling Space and Nonplanar Amino Groups on Free-Energy Predictions for RNA with Imino and Sheared Tandem GA Base Pairs Flanked by GC, CG, iGiC or iCiG Base Pairs

Guanine-adenine (GA) base pairs play important roles in determining the structure, dynamics, and stability of RNA. In RNA internal loops, GA base pairs often occur in tandem arrangements and their structure is context and sequence dependent. Calculations reported here test the thermodynamic integration (TI) approach with the amber99 force field by comparing computational predictions of free energy differences with the free energy differences expected on the basis of NMR determined structures of the RNA motifs (5′-GCG<u>GA</u>CGC-3′)₂, (5′-GCiG<u>GA</u>iCGC-3′)₂, (5′-GGC<u>GA</u>GCC-3′)₂, and (5′-GGiC<u>GA</u>iGCC-3′)₂. Here, iG and iC denote isoguanosine and isocytidine, which have amino and carbonyl groups transposed relative to guanosine and cytidine. The NMR structures show that the GA base pairs adopt either imino (<i>cis</i> Watson−Crick/Watson−Crick A-G) or sheared (<i>trans</i> Hoogsteen/Sugar edge A-G) conformations depending on the identity and orientation of the adjacent base pair. A new mixing function for the TI method is developed that allows alchemical transitions in which atoms can disappear in both the initial and final states. Unrestrained calculations gave Δ<i>G</i>° values 2−4 kcal/mol different from expectations based on NMR data. Restraining the structures with hydrogen bond restraints did not improve the predictions. Agreement with NMR data was improved by 0.7 to 1.5 kcal/mol, however, when structures were restrained with weak positional restraints to sample around the experimentally determined NMR structures. The amber99 force field was modified to partially include pyramidalization effects of the unpaired amino group of guanosine in imino GA base pairs. This provided little or no improvement in comparisons with experiment. The marginal improvement is observed when the structure has potential cross-strand out-of-plane hydrogen bonding with the G amino group. The calculations using positional restraints and a nonplanar amino group reproduce the signs of Δ<i>G</i>° from the experimental results and are, thus, capable of providing useful qualitative insights complementing the NMR experiments. Decomposition of the terms in the calculations reveals that the dominant terms are from electrostatic and interstrand interactions other than hydrogen bonds in the base pairs. The results suggest that a better description of the backbone is key to reproducing the experimental free energy results with computational free energy predictions

Controlled Oxidation of Remote sp³ C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity

The selective oxyfunctionalization of isolated sp³ C–H bonds in complex molecules represents a formidable challenge in organic chemistry. Here, we describe a rational, systematic strategy to expedite the development of P450 oxidation catalysts with refined regio- and stereoselectivity for the hydroxylation of remote, unactivated C–H sites in a complex scaffold. Using artemisinin as model substrate, we demonstrate how a three-tier strategy involving first-sphere active site mutagenesis, high-throughput P450 fingerprinting, and fingerprint-driven P450 reactivity predictions enabled the rapid evolution of three efficient biocatalysts for the selective hydroxylation of a primary and a secondary C–H site (with both <i>S</i> and <i>R</i> stereoselectivity) in a relevant yet previously inaccessible region of this complex natural product. The evolved P450 variants could be applied to provide direct access to the desired hydroxylated derivatives at preparative scales (0.4 g) and in high isolated yields (>90%), thereby enabling further elaboration of this molecule. As an example, enantiopure C7-fluorinated derivatives of the clinical antimalarial drugs artesunate and artemether, in which a major metabolically sensitive site is protected by means of a C–H to C–F substitution, were afforded via P450-mediated chemoenzymatic synthesis

Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics

The backbone dihedral parameters of the Amber RNA force field were improved by fitting using multiple linear regression to potential energies determined by quantum chemistry calculations. Five backbone and four glycosidic dihedral parameters were fit simultaneously to reproduce the potential energies determined by a high-level density functional theory calculation (B97D3 functional with the AUG-CC-PVTZ basis set). Umbrella sampling was used to determine conformational free energies along the dihedral angles, and these better agree with the population of conformations observed in the protein data bank for the new parameters than for the conventional parameters. Molecular dynamics simulations performed on a set of hairpin loops, duplexes and tetramers with the new parameter set show improved modeling for the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal loop of noncanonical pairs, as compared to the conventional parameters. For the tetramers, the new parameters largely avoid the incorrect intercalated structures that dominate the conformational samples from the conventional parameters. For the internal loop, the major conformation solved by NMR is stable with the new parameters, but not with the conventional parameters. The new force field performs similarly to the conventional parameters for the UUCG and GCAA hairpin loops and the [U­(UA)₆A]₂ duplex

Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics

The backbone dihedral parameters of the Amber RNA force field were improved by fitting using multiple linear regression to potential energies determined by quantum chemistry calculations. Five backbone and four glycosidic dihedral parameters were fit simultaneously to reproduce the potential energies determined by a high-level density functional theory calculation (B97D3 functional with the AUG-CC-PVTZ basis set). Umbrella sampling was used to determine conformational free energies along the dihedral angles, and these better agree with the population of conformations observed in the protein data bank for the new parameters than for the conventional parameters. Molecular dynamics simulations performed on a set of hairpin loops, duplexes and tetramers with the new parameter set show improved modeling for the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal loop of noncanonical pairs, as compared to the conventional parameters. For the tetramers, the new parameters largely avoid the incorrect intercalated structures that dominate the conformational samples from the conventional parameters. For the internal loop, the major conformation solved by NMR is stable with the new parameters, but not with the conventional parameters. The new force field performs similarly to the conventional parameters for the UUCG and GCAA hairpin loops and the [U­(UA)₆A]₂ duplex

Benchmarking AMBER Force Fields for RNA: Comparisons to NMR Spectra for Single-Stranded r(GACC) Are Improved by Revised χ Torsions

Accurately modeling unpaired regions of RNA is important for predicting structure, dynamics, and thermodynamics of folded RNA. Comparisons between NMR data and molecular dynamics simulations provide a test of force fields used for modeling. Here, NMR spectroscopy, including NOESY, ¹H–³¹P HETCOR, DQF-COSY, and TOCSY, was used to determine conformational preferences for single-stranded GACC RNA. The spectra are consistent with a conformational ensemble containing major and minor A-form-like structures. In a series of 50 ns molecular dynamics (MD) simulations with the AMBER99 force field in explicit solvent, initial A-form-like structures rapidly evolve to disordered conformations. A set of 50 ns simulations with revised χ torsions (AMBER99χ force field) gives two primary conformations, consistent with the NMR spectra. A single 1.9 μs MD simulation with the AMBER99χ force field showed that the major and minor conformations are retained for almost 68% of the time in the first 700 ns, with multiple transformations from A-form to non-A-form conformations. For the rest of the simulation, random-coil structures and a stable non-A-form conformation inconsistent with NMR spectra were seen. Evidently, the AMBER99χ force field improves structural predictions for single-stranded GACC RNA compared to the AMBER99 force field, but further force field improvements are needed

Single-Molecule Analysis of Cytochrome <i>c</i> Folding by Monitoring the Lifetime of an Attached Fluorescent Probe

Conformational dynamics of proteins are important for function. However, obtaining information about specific conformations is difficult for samples displaying heterogeneity. Here, time-resolved fluorescence resonance energy transfer is used to characterize the folding of single cytochrome <i>c</i> molecules. In particular, measurements of the fluorescence lifetimes of individual cytochrome <i>c</i> molecules labeled with a single dye that is quenched by energy transfer to the heme were used to monitor conformational transitions of the protein under partially denaturing conditions. These studies indicate significantly more conformational heterogeneity than has been described previously. Importantly, the use of a purified singly labeled sample made a direct comparison to ensemble data possible. The distribution of lifetimes of single proteins was compared to the distribution of lifetimes determined from analysis of ensemble lifetime fluorescence data. The results show broad agreement between single-molecule and ensemble data, with a similar range of lifetimes. However, the single-molecule data reveal greater conformational heterogeneity

Reparameterization of RNA χ Torsion Parameters for the AMBER Force Field and Comparison to NMR Spectra for Cytidine and Uridine

A reparameterization of the torsional parameters for the glycosidic dihedral angle, χ, for the AMBER99 force field in RNA nucleosides is used to provide a modified force field, AMBER99χ. Molecular dynamics simulations of cytidine, uridine, adenosine, and guanosine in aqueous solution using the AMBER99 and AMBER99χ force fields are compared with NMR results. For each nucleoside and force field, 10 individual molecular dynamics simulations of 30 ns each were run. For cytidine with AMBER99χ force field, each molecular dynamics simulation time was extended to 120 ns for convergence purposes. Nuclear magnetic resonance (NMR) spectroscopy, including one-dimensional (1D) ¹H, steady-state 1D ¹H nuclear Overhauser effect (NOE), and transient 1D ¹H NOE, was used to determine the sugar puckering and preferred base orientation with respect to the ribose of cytidine and uridine. The AMBER99 force field overestimates the population of syn conformations of the base orientation and of C2′-endo sugar puckering of the pyrimidines, while the AMBER99χ force field’s predictions are more consistent with NMR results. Moreover, the AMBER99 force field prefers high anti conformations with glycosidic dihedral angles around 310° for the base orientation of purines. The AMBER99χ force field prefers anti conformations around 185°, which is more consistent with the quantum mechanical calculations and known 3D structures of folded ribonucleic acids (RNAs). Evidently, the AMBER99χ force field predicts the structural characteristics of ribonucleosides better than the AMBER99 force field and should improve structural and thermodynamic predictions of RNA structures

Revision of AMBER Torsional Parameters for RNA Improves Free Energy Predictions for Tetramer Duplexes with GC and iGiC Base Pairs

All-atom force fields are important for predicting thermodynamic, structural, and dynamic properties of RNA. In this paper, results are reported for thermodynamic integration calculations of free energy differences of duplex formation when CG pairs in the RNA duplexes r(CCGG)₂, r(GGCC)₂, r(GCGC)₂, and r(CGCG)₂ are replaced by isocytidine–isoguanosine (iCiG) pairs. Agreement with experiment was improved when ε/ζ, α/γ, β, and χ torsional parameters in the AMBER99 force field were revised on the basis of quantum mechanical calculations. The revised force field, AMBER99TOR, brings free energy difference predictions to within 1.3, 1.4, 2.3, and 2.6 kcal/mol at 300 K, respectively, compared to experimental results for the thermodynamic cycles of CCGG → iCiCiGiG, GGCC → iGiGiCiC, GCGC → iGiCiGiC, and CGCG → iCiGiCiG. In contrast, unmodified AMBER99 predictions for GGCC → iGiGiCiC and GCGC → iGiCiGiC differ from experiment by 11.7 and 12.6 kcal/mol, respectively. In order to test the dynamic stability of the above duplexes with AMBER99TOR, four individual 50 ns molecular dynamics (MD) simulations in explicit solvent were run. All except r(CCGG)₂ retained A-form conformation for ≥82% of the time. This is consistent with NMR spectra of r(iGiGiCiC)₂, which reveal an A-form conformation. In MD simulations, r(CCGG)₂ retained A-form conformation 52% of the time, suggesting that its terminal base pairs may fray. The results indicate that revised backbone parameters improve predictions of RNA properties and that comparisons to measured sequence dependent thermodynamics provide useful benchmarks for testing force fields and computational methods